18-Membered Macrocycles and Analogs Thereof

ABSTRACT

The present invention relates generally to the 18-membered macrocyclic antimicrobial agents called Tiacumicins, specifically, OPT-80 (which is composed almost entirely of the R-Tiacumicin B), pharmaceutical compositions comprising OPT-80, and methods using OPT-80. In particular, this compound is a potent drug for the treatment of bacterial infections, specifically  C. difficile  infections.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part application ofInternational Application PCT/US2005/002887, filed Jan. 31, 2005, whichis incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates generally to the 18-membered macrocyclicantimicrobial agents called Tiacumicins, specifically, the R-TiacumicinB or Tiacumicin B and its related compounds. In particular,substantially pure R-Tiacumicin B, as a potent antibiotic agent for thetreatment of bacterial infections, specifically GI infections caused bytoxin producing strains of Clostridium difficile (C. difficile),Staphylococcus aureus (S. aureus) including methicillin-resistantStaphylococcus aureus (MRSA) and Clostridium perfringens (C.perfringens).

BACKGROUND OF THE INVENTION

Macrocycles are an important therapeutic class of antibiotics. Thesecompounds are frequently produced as a family of closely relatedbiogenetic congeners. The Tiacumicins are a series of 18-memberedmacrocyclic antibiotics in which the macrocyclic ring is glycosidicallyattached to one or two sugars. A seven-carbon sugar is esterfied atvarious positions with small fatty acids. The other sugar, when present,is esterified with an isomer of the fully substituted benzoic acid,everninic acid. (Journal of Liquid Chromatography, 1988, 11: 191-201).

Tiacumicins are a family of related compounds that contain the18-membered ring shown in Formula I below.

At present, several distinct Tiacumicins have been identified and six ofthese (Tiacumicin A-F) are defined by their particular pattern ofsubstituents R¹, R², and R³ (U.S. Pat. No. 4,918,174; J. Antibiotics,1987, 40: 575-588), as shown in Table 1.

TABLE 1 Substituents Present In Tiacumcins A-F R¹ R² R³ A

H H B

OH C

OH D

OH E

OH F

OH

Tiacumicins A-F have been characterized spectroscopically and by otherphysical methods. The chemical structures of Tiacumicins are based onspectroscopy: UV-vis, IR and ¹H and ¹³C NMR, see for example J.Antibiotics, 1987, 40: 575-588. Inspection of Table 1 reveals thatcertain members of the family are structurally related isomers and/ordiffer by the presence or absence of certain moieties. Others differ inthe nature of their ester groups.

Tiacumicins are produced by bacteria, including Dactylosporangiumaurantiacum subspecies hamdenensis, which may be obtained from the ARSPatent Collection of the Northern Regional Research Center, UnitedStates Department of Agriculture, 1815 North University Street, Peoria,Ill. 61604, accession number NRRL 18085. The characteristics of strainAB 718C-41 are given in J. Antibiotics, 1987, 40: 567-574 and U.S. Pat.No. 4,918,174.

C. difficile-associated diarrhea (CDAD) is a disease characterized bysevere and painful diarrhea. C. difficile is responsible forapproximately 20% of the cases of antibiotic-associated diarrhea (AAD)and the majority of the cases of antibiotic-associated colitis (AAC).These diseases are typically caused by toxin producing strains of C.difficile, S. aureus including methicillin-resistant S. aureus (MRSA)and Clostridium perfringens (C. perfringens). AAD represents a majoreconomic burden to the healthcare system that is conservativelyestimated at $3-6 billion per year in excess hospital costs in the U.S.alone.

Vancomycin-resistant enterococci, for which intestinal colonizationprovides a constant reservoir for infection, has also emerged as a majornosocomial pathogen associated with increased health care cost andmortality. VRE can appear as coinfection in patients infected with C.difficile, or more commonly cause infection in certain high riskpatients such as haematology and oncology patients, patients inintensive care units and patients receiving solid organ transplants.

Methicillin-resistant Staphylococci, such as MRSA, are increasing inprevalence in both the hospital and community settings. Staphylococciare found on the skin and within the digestive and respiratory tractsbut can infect open wounds and burns and can progress to serioussystemic infection. The emergence of multi-drug resistant Staphylococci,especially, in the hospital where antibiotic use is frequent andselective pressure for drug-resistant organisms is high, has proven achallenge for treating these patients. The presence of MRSA on the skinof patients and health care workers promotes transmission of themulti-drug resistant organisms.

Similar diseases, including but not limited to clostridialenterocolitis, neonatal diarrhea, antibiotic-associated enterocolitis,sporadic enterocolitis, and nosocomial enterocolitis are alsosignificant problems in some animal species.

AAD is a significant problem in hospitals and long-term care facilitiesand in the community. C. difficile is the leading cause of AAD in thehospital setting, accounting for approximately 20% of cases of AAD andthe majority of cases of antibiotic-associated colitis (AAC). The risingincidence of Clostridium difficile-associated diarrhea (CDAD) has beenattributed to the frequent prescription of broad-spectrum antibiotics tohospitalized patients.

The most serious form of the disease is pseudomembranous colitis (PMC),which is manifested histologically by colitis with mucosal plaques, andclinically by severe diarrhea, abdominal cramps, and systemic toxicity.The overall mortality rate from CDAD is low, but is much greater inpatients who develop severe colitis or systemic toxicity. A recent studyhas shown that even when death is not directly attributable to C.difficile, the rate of mortality in CDAD patients as compared tocase-matched controls is much greater.

Diarrhea and colitis are caused by the elaboration of one or more C.difficile toxins. The organism proliferates in the colon in patients whohave been given broad-spectrum antibiotics or, less commonly, cancerchemotherapy. CDAD is diagnosed in approximately 20% of hospitalizedpatients who develop diarrhea after treatment with such agents.

There are currently two dominant therapies for CDAD: vancomycin andmetronidazole. Vancomycin is not recommended for first-line treatment ofCDAD mainly because it is the only antibiotic active against someserious life-threatening multi-drug resistant bacteria. Therefore, in aneffort to minimize the emergence of vancomycin-resistant Enterococcus(VRE) or vancomycin-resistant S. aureus (VRSA), the medical communitydiscourages the use of this drug except when absolutely necessary.

Metronidazole is recommended as initial therapy out of concern for thepromotion and selection of vancomycin resistant gut flora, especiallyenterococci. Despite reports that the frequency of C. difficileresistance may be >6% in some countries, metronidazole remains nearly aseffective as vancomycin, is considerably less expensive, and can be usedeither orally or intravenously. Metronidazole is associated withsignificant adverse effects including nausea, neuropathy, leukopenia,seizures, and a toxic reaction to alcohol. Furthermore, it is not safefor use in children or pregnant women. Clinical recurrence occurs in upto 20% of cases after treatment with either vancomycin or metronidazole.Therapy with metronidazole has been reported to be an important riskfactor for VRE colonization and infection. The current treatment regimeagainst Gastrointestinal infections, e.g., Clostridiumdifficile-associated diarrhea (CDAD) is rather cumbersome, requiring upto 500 mg four-times daily for 10 to 14 days. Thus, there is a need forbetter treatment for cases of CDAD as well as for cases of otherAntibiotic-associated diarrhea (AAD) and Antibiotic-associated colitis(AAC).

Tiacumicins, specifically Tiacumicin B, show activity against a varietyof bacterial pathogens and in particular against C. difficile, aGram-positive bacterium (Antimicrob. Agents Chemother. 1991, 1108-1111).C. difficile is an anaerobic spore-forming bacterium that causes aninfection of the bowel. Diarrhea is the most common symptom butabdominal pain and fever may also occur. C. difficile is a majorcausative agent of colitis (inflammation of the colon) and diarrhea thatmay occur following antibiotic intake. This bacterium is primarilyacquired in hospitals and chronic care facilities. Because Tiacumicin Bshows promising activity against C. difficile, it is expected to beuseful in the treatment of bacterial infections, especially those of thegastrointestinal tract, in mammals. Examples of such treatments includebut are not limited to treatment of colitis and treatment of irritablebowel syndrome. Tiacumicins may also find use for the treatment ofgastrointestinal cancers.

Tiacumicin antibiotics are described in U.S. Pat. No. 4,918,174 (issuedApr. 17, 1990), J. Antibiotics 1987, 40: 575-588, J. Antibiotics 1987,40: 567-574, J. Liquid Chromatography 1988, 11: 191-201, AntimicrobialAgents and Chemotherapy 1991, 35: 1108-1111, U.S. Pat. No. 5,583,115(issued Dec. 10, 1996), and U.S. Pat. No. 5,767,096 (issued Jun. 16,1998), which are all incorporated herein by reference. Related compoundsare the Lipiarmycin antibiotics (c.f., J. Chem. Soc. Perkin Trans. I,1987, 1353-1359 and J. Antibiotics 1988, 41: 308-315) and theClostomicin antibiotics (J. Antibiotics 1986, 39: 1407-1412), which areall incorporated herein by reference.

SUMMARY OF THE INVENTION

The present invention relates to new pharmaceutical compositionscontaining R-Tiacumicins, specifically the optically pure R-TiacumicinB, and to the use of these new compositions in combination with existingdrugs to treat infections caused by gram-positive anerobes.

One embodiment of the present invention is directed towards thediscovery that the chiral center at C-19 of Tiacumicin B has greateffect on biological activity. It has now been discovered that asubstantially pure preparation of higher activity R-Tiacumicin B, whichhas an R-hydroxy group at C-19 has surprisingly lower MIC values thanthe optically pure S-isomer of Tiacumicin B and other Tiacumicin Brelated compounds.

In another embodiment of the present invention the substantially pureR-Tiacumicin B has an unusually long post-antibiotic activity (PAE).

This invention encompasses the composition of novel antibiotic agents,containing substantially pure R-Tiacumicins, by submerged aerobicfermentation of the microorganism Dactylosporangium aurantiacumsubspecies hamdenensis. The production method is covered by WO2004/014295 A2, which is hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the Oak Ridge Thermal Ellipsoid Plot Program (ORTEP)chemical structure of R-Tiacumicin B.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term “antibiotic-associated condition” refers to a conditionresulting when antibiotic therapy disturbs the balance of the microbialflora of the gut, allowing pathogenic organisms such as enterotoxinproducing strains of C. difficile, S. aureus and C. perfringens toflourish. These organisms can cause diarrhea, pseudomembranous colitis,and colitis and are manifested by diarrhea, urgency, abdominal cramps,tenesmus, and fever among other symptoms. Diarrhea, when severe, causesdehydration and the medical complications associated with dehydration.

The term “asymmetrically substituted” refers to a molecular structure inwhich an atom having four tetrahedral valences is attached to fourdifferent atoms or groups. The commonest cases involve the carbon atom.In such cases, two optical isomers (D- and L-enantiomers or R— and S—enantiomers) per carbon atom result which are nonsuperposable mirrorimages of each other. Many compounds have more than one asymmetriccarbon. This results in the possibility of many optical isomers, thenumber being determined by the formula 2^(n), where n is the number ofasymmetric carbons.

As used herein, and unless otherwise indicated, the terms“biohydrolyzable carbamate,” “biohydrolyzable carbonate,”“biohydrolyzable ureide” and “biohydrolyzable phosphate” mean acarbamate, carbonate, ureide and phosphate, respectively, of a compoundthat either: 1) does not interfere with the biological activity of thecompound but can confer upon that compound advantageous properties invivo, such as uptake, duration of action, or onset of action; or 2) isbiologically inactive but is converted in vivo to the biologicallyactive compound. Examples of biohydrolyzable carbamates include, but arenot limited to, lower alkylamines, substituted ethylenediamines,aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines,and polyether amines.

As used herein, and unless otherwise indicated, the term“biohydrolyzable ester” means an ester of a compound that either: 1)does not interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable esters include, but are not limited to,lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters,and choline esters.

As used herein, and unless otherwise indicated, the term“biohydrolyzable amide” means an amide of a compound that either: 1)does not interfere with the biological activity of the compound but canconfer upon that compound advantageous properties in vivo, such asuptake, duration of action, or onset of action; or 2) is biologicallyinactive but is converted in vivo to the biologically active compound.Examples of biohydrolyzable amides include, but are not limited to,lower alkyl amides, .alpha.-amino acid amides, alkoxyacyl amides, andalkylaminoalkylcarbonyl amides.

The term “broth” as used herein refers to the fluid culture medium asobtained during or after fermentation. Broth comprises a mixture ofwater, the desired antibiotic(s), unused nutrients, living or deadorganisms, metabolic products, and the adsorbent with or withoutadsorbed product.

The term “C-19 Ketone” refers to a Tiacumicin B related compound shownbelow in Formula II:

The term “diastereomers” refers to stereoisomers that are not mirrorimages of each other.

The term “enantiomer” refers to a non-superimposable mirror image ofitself. An enantiomer of an optically active isomer rotates planepolarized light in an equal but opposite direction of the originalisomer. A solution of equal parts of an optically active isomer and itsenantiomer is known as a racemic solution and has a net rotation ofplane polarized light of zero. Enantiomers will have the oppositeprefixes of each other: D- becomes L- or R— becomes S—. Often only oneenantiomer is active in a biological system, because most biologicalreactions are enzymatic and the enzymes can only attach to one of theenantiomers.

The term “excipient” refers to an inert substance added to apharmacological composition to further facilitate administration of acompound. Examples of excipients include but are not limited to, calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

The term “halogen” includes F, Cl, Br and I.

As used herein, the term “hydrate” means a compound of the presentinvention or a salt thereof that further includes a stoichiometric ornon-stoichiometric amount of water bound by non-covalent intermolecularforces.

The term “isomeric mixture” means a mixture of two or moreconfigurationally distinct chemical species having the same chemicalformula. An isomeric mixture is a genus comprising individual isomericspecies. Examples of isomeric mixtures include stereoisomers(enantiomers and diastereomers), regioisomers, as might result forexample from a pericyclic reaction. The compounds of the presentinvention comprise asymmetrically substituted carbon atoms. Suchasymmetrically substituted carbon atoms can result in mixtures ofstereoisomers at a particular asymmetrically substituted carbon atom ora single stereoisomer. As a result, racemic mixtures, mixtures ofdiastereomers, as well as single diastereomers of the compounds of theinvention are included in the present invention.

The term “Lipiarmycin A4” refers to a Tiacumicin B related compoundshown below in Formula III:

The term “lower alkyl,” alone or in combination, refers to an optionallysubstituted straight-chain or optionally substituted branched-chainhaving from 1 to about 8 carbons (e.g., C₁, C₂, C₃, C₄, C₅, C₆, C₇,C₈,), more preferably 1 to 4 carbons (e.g., C₁, C₂, C₃, C₄,). Examplesof alkyl radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, tert-butyl. A “lower alkyl” is generally a shorteralkyl, e.g., one containing from 1 to about 4 carbon atoms (e.g., C₁,C₂, C₃, C₄,).

The term “macrocycles” refers to organic molecules with large ringstructures usually containing over 10 atoms.

The term “18-membered macrocycles” refers to organic molecules with ringstructures containing 18 atoms.

The term “membered ring” can embrace any cyclic structure, includingcarbocycles and heterocycles as described above. The term “membered” ismeant to denote the number of skeletal atoms that constitute the ring.Thus, for example, pyridine, pyran and thiopyran are 6 membered ringsand pyrrole, furan, and thiophene are 5 membered rings.

The term “MIC” or “minimum inhibitory concentration” refers to thelowest concentration of an antibiotic that is needed to inhibit growthof a bacterial isolate in vitro. A common method for determining the MICof an antibiotic is to prepare several tubes containing serial dilutionsof the antibiotic, that are then inoculated with the bacterial isolateof interest. The MIC of an antibiotic can be determined from the tubewith the lowest concentration that shows no turbidity (no growth).

The term “MIC₅₀” refers to the lowest concentration of antibioticrequired to inhibit the growth of 50% of the bacterial strains testedwithin a given bacterial species.

The term “MIC₉₀” refers to the lowest concentration of antibioticrequired to inhibit the growth of 90% of the bacterial strains testedwithin a given bacterial species.

The term “OPT-80” refers to a preparation containing R-Tiacumicin B andTiacumicin B related compounds (including, but not limited to,Tiacumicins, Lipiarmycin A4 and C-19 Ketone). Preparations of this typeare described in detail in PCT application PCT/US03/21977, having aninternational publication number of WO 2004/014295 A2 and whichpreparations and are incorporated here by reference.

The term “ORTEP” refers to the Oak Ridge Thermal Ellipsoid Plot computerprogram, written in Fortran, for drawing crystal structureillustrations. Ball-and-stick type illustrations of a quality suitablefor publication are produced with either spheres or thermal-motionprobability ellipsoids, derived from anisotropic temperature factorparameters, on the atomic sites. The program also produces stereoscopicpairs of illustrations which aid in the visualization of complexarrangements of atoms and their correlated thermal motion patterns.

The term “PAE” or “post-antibiotic effect” refers to a well-establishedpharmacodynamic parameter that reflects the persistent suppression ofbacterial growth following antibiotic exposure.

The term “patient” refers to a human or animal in need of medicaltreatment. For the purposes of this invention, human patients aretypically institutionalized in a primary medical care facility such as ahospital or nursing home. However, treatment of a disease associatedwith the use of antibiotics or cancer chemotherapies or antiviraltherapies can occur on an outpatient basis, upon discharge from aprimary care facility, or can be prescribed by a physician forhome-care, not in association with a primary medical care facilityAnimals in need of medical treatment are typically in the care of aveterinarian.

The term “pharmaceutically acceptable carrier” refers to a carrier ordiluent that is pharmaceutically acceptable.

The term “pharmaceutically acceptable salts” refers to those derivedfrom pharmaceutically acceptable inorganic and organic bases. Saltsderived from appropriate bases include alkali metal (e.g., sodium orpotassium), alkaline earth metal (e.g., magnesium), ammonium and N(C₁-C₄alkyl)₄ ⁺ salts, and the like. Illustrative examples of some of theseinclude sodium hydroxide, potassium hydroxide, choline hydroxide, sodiumcarbonate, and the like. The term “pharmaceutically acceptable salt”also refers to salts prepared from pharmaceutically acceptable non-toxicacids, including inorganic acids and organic acids. Suitable non-toxicacids include inorganic and organic acids such as, but not limited to,acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic,citric, ethenesulfonic, formic, fumaric, furoic, gluconic, glutamic,glucorenic, galacturonic, glycidic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phenylacetic, propionic, phosphoric,salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid,p-toluenesulfonic and the like. Particularly preferred are hydrochloric,hydrobromic, phosphoric, and sulfuric acids, and most particularlypreferred is the hydrochloride salt.

The term “pharmaceutical composition” refers to a composition of theR-Tiacumicin described herein, or physiologically acceptable saltsthereof, with other chemical components, such as physiologicallyacceptable carriers and/or excipients. The purpose of a pharmaceuticalcomposition is to facilitate administration of a compound to a mammal,including humans.

The term “physiologically acceptable carrier” refers to a carrier ordiluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered compound.

As used herein, and unless otherwise indicated, the term “prodrug” meansa derivative of a compound that can hydrolyze, oxidize, or otherwisereact under biological conditions (in vitro or in vivo) to provide thecompound. Examples of prodrugs include, but are not limited to,compounds that comprise biohydrolyzable moieties such as biohydrolyzableamides, biohydrolyzable esters, biohydrolyzable carbamates,biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzablephosphate analogues. Other examples of prodrugs include compounds thatcomprise —NO, —NO₂, —ONO, or —ONO₂ moieties. When used to describe acompound of the invention, the term “prodrug” may also to be interpretedto exclude other compounds of the invention for example racemates.

The term “pseudomembranous colitis” or “enteritis” refers to theformation of pseudomembranous material (i.e., material composed offibrin, mucous, necrotic epithelial cells and leukocytes) due toinflammation of the mucous membrane of both the small and largeintestine.

The terms “R” and “S” configuration, as used herein, are as defined bythe IUPAC 1974 Recommendations for Section E, FundamentalStereochemistry, Pure Appl. Chem. (1976) 45, 13-30. Chiral molecules canbe named based on the atomic numbers of the atoms or groups of atoms,the ligands that are attached to the chiral center. The ligands aregiven a priority (the higher the atomic number the higher the priority)and if the priorities increase in a clockwise direction, they are saidto be R—. Otherwise, if they are prioritized in a counterclockwisedirection they are said to be S—.

The term “R-Tiacumicin B” refers to the optically pure (R)-isomer ofTiacumicin B with an (R)-hydroxy group at C-19, as shown below inFormula IV:

The term “S-Tiacumicin B” refers to the optically pure (S)-isomer ofTiacumicin B with an (S)-hydroxy group at C-19, as shown below inFormula V:

The term “stereoisomers” refers to compounds whose molecules have thesame number and kind of atoms and the same atomic arrangement, butdiffer in their spatial arrangement.

As used herein, and unless otherwise indicated, the terms “opticallypure,” “stereomerically pure,” and “substantially stereomerically pure”are used interchangeably and mean one stereoisomer of a compound or acomposition that comprises one stereoisomer of a compound and issubstantially free of other stereoisomer(s) of that compound. Forexample, a stereomerically pure compound or composition of a compoundhaving one chiral center will be substantially free of the oppositeenantiomer of the compound. A stereomerically pure compound orcomposition of a compound having two chiral centers will besubstantially free of other diastereomers of the compound. A typicalstereomerically pure compound comprises greater than about 80% by weightof one stereoisomer of the compound and less than about 20% by weight ofother stereoisomers of the compound, more preferably greater than about90% by weight of one stereoisomer of the compound and less than about10% by weight of the other stereoisomers of the compound, even morepreferably greater than about 95% by weight of one stereoisomer of thecompound and less than about 5% by weight of the other stereoisomers ofthe compound, and most preferably greater than about 97% by weight ofone stereoisomer of the compound and less than about 3% by weight of theother stereoisomers of the compound.

The term “sugar” generally refers to mono-, di- or oligosaccharides. Asaccharide may be substituted, for example, glucosamine, galactosamine,acetylglucose, acetylgalactose, N-acetylglucosamine,N-acetyl-galactosamine, galactosyl-N-acetylglucosamine,N-acetylneuraminic acid (sialic acid), etc., as well as sulfated andphosphorylated sugars. For the purposes of this definition, thesaccharides are in their pyranose or furanose form.

The term “Tiacumicin” as used herein refers to a family of compounds allof which comprise the 18-membered macrocycle shown below in Formula I:

The term “Tiacumicin B” as used herein refers to the 18-memberedmacrocycle shown below in Formula VI:

The term “yield” as used herein refers to an amount of crude Tiacumicinre-constituted in methanol to the same volume as the originalfermentation broth. Yield is determined using standard HPLC techniques.Yield is reported in units of mg/L.

This invention encompasses the composition of novel antibiotic agents,Tiacumicins, by submerged aerobic fermentation of the microorganismDactylosporangium aurantiacum subspecies hamdenensis. The productionmethod is covered by WO 2004/014295 A2.

The present invention relates to new antibacterial compositionscontaining R-Tiacumicins, specifically the R-Tiacumicin B (which has anR-hydroxyl at C-19), and to the use of these new compositions incombination with existing drugs to treat infections caused bygram-positive anerobes.

The present invention further relates to stereoisomerically pureTiacumicin B, which contains 90-100% of the R-stereoisomer, preferablyat least 93% of the R-stereoisomer, more preferably 95% of theR-stereoisomer, even more preferably 99% of the R-stereoisomer.

In accordance with the present invention there are provided compoundswith the structure of Formula VII:

wherein:X is selected from lower alkyl, and wherein the term “lower alkyl” asused herein refers to branched or straight chain alkyl groups comprisingone to two carbon atoms, including methyl, ethyl, n-propyl, isopropyl,and the like; andY is selected from OH or a ketone (═O); andZ is selected from H or lower alkyl, and wherein the term “lower alkyl”as used herein refers to branched or straight chain alkyl groupscomprising one to five carbon atoms, including methyl, ethyl, propyl,isopropyl, n-butyl, t-butyl, and the like.

Preferred compounds of the invention are compounds of Formula VIIwherein X is methyl or ethyl, Y is ketone (═O) or OH and Z is isopropyl.

More preferred compounds of the invention are the compound of theFormula VII wherein X is ethyl, Y is ketone (═O) or OH and Z isisopropyl.

The most preferred compounds of the invention are the compounds ofFormula VII wherein X is ethyl, Y is OH R and Z is isopropyl.

One embodiment of the present invention is directed towards thediscovery that the chiral center at C-19 of Tiacumicin B has greateffect on biological activity. It has now been discovered thatR-Tiacumicin B, which has an R-hydroxy group at C-19 has significantlyhigher activity than the S-Tiacumicin B and other Tiacumicin B relatedcompounds (Lipiarmycin A4 and C-19 Ketone). The higher activity is shownby much lowered MIC values, which can be seen below in Example 3, Tables3 and 4 for several strains of C. difficile, S. aureus, E. faecalis, andE. faecium. This effect of the C-19 chiral center on biological activityis an unexpected and novel discovery.

In another embodiment of the present invention OPT-80 (which is composedalmost entirely of the R-Tiacumicin B) has an unusually longpost-antibiotic effect (PAE). This is discussed below in Example 4,where it is shown that OPT-80 has a PAE of greater than 24 hours. ThisPAE is unexpectedly longer than the usual antibiotic PAE of 1-5 hours.

The present invention also relates to the disclosure of pharmaceuticalcompositions, which comprise a compound of the present invention incombination with a pharmaceutically acceptable carrier.

Yet another aspect of the invention discloses a method of inhibiting ortreating bacterial infections in humans, comprising administering to thepatient a therapeutically effective amount of a compound of theinvention alone or in combination with another antibacterial orantifungal agent.

Production

The 18-membered macrocycles and analogs thereof are produced byfermentation. Cultivation of Dactylosporangium aurantiacum subsp.hamdenensis AB 718C-41 NRRL 18085 for the production of the Tiacumicinsis carried out in a medium containing carbon sources, inorganic saltsand other organic ingredients with one or more absorbents under properaeration conditions and mixing in a sterile environment.

The microorganism to produce the active antibacterial agents wasidentified as belonging to the family Actinoplanaceae, genusDactylosporangium (J. of Antibiotics, 1987, 40: 567-574 and U.S. Pat.No. 4,918,174). It has been designated Dactylasporangium aurantiacumsubspecies hamdenensis 718C-41. The subculture was obtained from the ARSPatent Collection of the Northern Regional Research Center, UnitedStates Department of Agriculture, 1815 North University Street, Peoria,Ill. 61604, U.S.A., where it was assigned accession number NRRL 18085.The characteristics of strain AB 718C-41 are given in the Journal ofAntibiotics, 1987, 40: 567-574 and U.S. Pat. No. 4,918,174.

Methods of isolating stereomerically pure isomers are known in the art.Methods of isolating stereomerically pure R-Tiacumicin include, but arenot limited to, recrystallization of the crude mixture in solventsincluding, aqueous methanol or isopropanol and chiral HPLC.

This invention encompasses the composition of novel antibiotic agents,Tiacumicins, by submerged aerobic fermentation of the microorganismDactylosporangium aurantiacum subspecies hamdenensis. The productionmethod is covered by WO 2004/014295 A2, which is hereby incorporated byreference.

Pharmaceutical Formulation and Administration

Pharmaceutical compositions of the Tiacumicin compounds of the presentinvention, specifically OPT-80 (which is composed almost entirely of theR-Tiacumicin), according to the invention may be formulated to releasean antibiotic substantially immediately upon administration or at anypredetermined time or time period after administration.

The latter types of compositions are generally known as modified releaseformulations, which include formulations that create a substantiallyconstant concentration of the drug within the intestinal tract over anextended period of time, and formulations that have modified releasecharacteristics based on temporal or environmental criteria as describedin Modified-Release Drug Delivery Technology, ed. M. J. Rathbone, J.Hodgraft and M. S. Roberts. Marcel Dekker, Inc. New York.

Any oral biologically-acceptable dosage form, or combinations thereof,can be employed in the methods of the invention. Examples of such dosageforms include, without limitation, chewable tablets, quick dissolvetablets, effervescent tablets, reconstitutable powders, elixirs,liquids, suppositories, creams, solutions, suspensions, emulsions,tablets, multi-layer tablets, bi-layer tablets, capsules, soft gelatincapsules, hard gelatin capsules, osmotic tablets, osmotic capsules,caplets, lozenges, chewable lozenges, beads, powders, granules,particles, microparticles, dispersible granules, ingestibles, infusions,health bars, confections, animal feeds, cereals, cereal coatings, foods,nutritive foods, functional foods and combinations thereof. Thepreparation of any of the above dosage forms is well known to persons ofordinary skill in the art. Additionally, the pharmaceutical formulationsmay be designed to provide either immediate or controlled release of theantibiotic upon reaching the target site. The selection of immediate orcontrolled release compositions depends upon a variety of factorsincluding the species and antibiotic susceptibility of Gram-positivebacteria being treated and the bacteriostatic/bactericidalcharacteristics of the therapeutics. Methods well known in the art formaking formulations are found, for example, in Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, 2000, LippincottWilliams & Wilkins, Philadelphia, or in Encyclopedia of PharmaceuticalTechnology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, MarcelDekker, New York.

Immediate release formulations for oral use include tablets or capsulescontaining the active ingredient(s) in a mixture with non-toxicpharmaceutically acceptable excipients. These excipients may be, forexample, inert diluents or fillers (e.g., sucrose, sorbitol, sugar,mannitol, microcrystalline cellulose, starches including potato starch,calcium carbonate, sodium chloride, lactose, calcium phosphate, calciumsulfate, or sodium phosphate); granulating and disintegrating agents(e.g., cellulose derivatives including microcrystalline cellulose,starches including potato starch, croscarmellose sodium, alginates, oralginic acid); binding agents (e.g., sucrose, glucose, mannitol,sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch,pregelatinized starch, microcrystalline cellulose, magnesium aluminumsilicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like as are found, for example, in The Handbook ofPharmaceutical Excipients, third edition, edited by Arthur H. Kibbe,American Pharmaceutical Association Washington D.C.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

A controlled release composition may also be in the form of a buoyanttablet or capsule (i.e., a tablet or capsule that, upon oraladministration, floats on top of the gastric content for a certainperiod of time). A buoyant tablet formulation of the compound(s) can beprepared by granulating a mixture of the antibiotic with excipients and20-75% w/w of hydrocolloids, such as hydroxyethylcellulose,hydroxypropylcellulose, or hydroxypropyl-methylcellulose. The obtainedgranules can then be compressed into tablets. On contact with thegastric juice, the tablet forms a substantially water-impermeable gelbarrier around its surface. This gel barrier takes part in maintaining adensity of less than one, thereby allowing the tablet to remain buoyantin the gastric juice. Other useful controlled release compositions areknown in the art (see, for example, U.S. Pat. Nos. 4,946,685 and6,261,601).

A modified release composition may be comprised of a compression-coatedcore whose geometric configuration controls the release profile of theencapsulated antibiotic. By varying the geometry of the core, theprofile of the antibiotic release can be adjusted to follow zero order,first order or a combination of these orders. The system can also bedesigned to deliver more beneficial agents at the same time, each havinga different release profile (see, for example U.S. Pat. Nos. 4,111,202and 3,279,995).

Formulations that target the Tiacumicin compounds of the presentinvention, specifically OPT-80 (which is composed almost entirely of theR-Tiacumicin), that release to particular regions of the intestinaltract can also be prepared. The Tiacumicin compounds of the presentinvention, specifically OPT-80, can be encapsulated in an entericcoating that prevents release degradation and release from occurring inthe stomach, but dissolves readily in the mildly acidic or neutral pHenvironment of the small intestine. A formulation targeted for releaseof antibiotic to the colon, utilizing technologies such astime-dependent, pH-dependent, or enzymatic erosion of polymer matrix orcoating can also be used.

The targeted delivery properties of the Tiacumicin compounds of thepresent invention, specifically OPT-80 (which is composed almostentirely of the R-Tiacumicin B), containing formulation may be modifiedby other means. For example, the antibiotic may be complexed byinclusion, ionic association, hydrogen bonding, hydrophobic bonding, orcovalent bonding. In addition polymers or complexes susceptible toenzymatic or microbial lysis may also be used as a means to deliverdrug.

Microsphere encapsulation of the Tiacumicin compounds of the presentinvention, specifically OPT-80 (which is composed almost entirely of theR-Tiacumicin B), is another useful pharmaceutical formulation fortargeted antibiotic release. The antibiotic-containing microspheres canbe used alone for antibiotic delivery, or as one component of atwo-stage release formulation. Suitable staged release formulations mayconsist of acid stable microspheres, encapsulating the compounds of thepresent invention, specifically OPT-80 (which is composed almostentirely of the R-Tiacumicin B), to be released later in the lowerintestinal tract admixed with an immediate release formulation todeliver antibiotic to the stomach and upper duodenum.

Microspheres can be made by any appropriate method, or from anypharmaceutically acceptable material. Particularly useful are proteinoidmicrospheres (see, for example, U.S. Pat. No. 5,601,846, or 5,792,451)and PLGA-containing microspheres (see, for example, U.S. Pat. No.6,235,224 or 5,672,659). Other polymers commonly used in the formationof microspheres include, for example, poly-ε-caprolactone,poly(e˜caprolactone-Co-DL-lactic acid), poly(DL-lactic acid),poly(DL-lactic acid-Co-glycolic acid) andpoly(s-caprolactone-Co-glycolic acid) (see, for example, Pitt et al., J.Pharm. Sci., 68:1534, 1979). Microspheres can be made by procedures wellknown in the art including spray drying, coacervation, andemulsification (see for example Davis et al. Microsphere and DrugTherapy, 1984, Elsevier; Benoit et al. Biodegradable Microspheres:Advances in Production Technologies, Chapter 3, ed. Benita, S, 1996,Dekker, New York; Microencapsulation and Related Drug Processes, Ed.Deasy, 1984, Dekker, New York; U.S. Pat. No. 6,365,187).

Powders, dispersible powders, or granules suitable for preparation ofaqueous solutions or suspensions of the Tiacumicin compounds of thepresent invention, specifically OPT-80 (which is composed almostentirely of the R-Tiacumicin B), by addition of water are convenientdosage forms for oral administration. Formulation as a suspensionprovides the active ingredient in a mixture with a dispersing or wettingagent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents are, for example, naturally-occurringphosphatides (e.g., lecithin or condensation products of ethylene oxidewith a fatty acid, a long chain aliphatic alcohol, or a partial esterderived from fatty acids) and a hexitol or a hexitol anhydride (e.g.,polyoxyethylene stearate, polyoxyethylene sorbitol monooleate,polyoxyethylene sorbitan monooleate, and the like). Suitable suspendingagents are, for example, sodium carboxymethylcellulose, methylcellulose,sodium alginate, and the like.

EXAMPLES

The following examples are provided by way of describing specificembodiments of the present invention without intending to limit thescope of the invention in any way.

Example 1 Exact Structure of R-Tiacumicin B

The exact structure of the R-Tiacumicin B (the major most activecomponent of OPT-80) is shown below in Formula IV. The X-ray crystalstructure of the R-Tiacumicin B was obtained from a colorless,parallelepiped-shaped crystal (0.08×0.14×0.22 mm) grown in methanol andis shown as an ORTEP diagram in FIG. 1. This x-ray structure confirmsthe structure shown below in Formula IV. The official chemical name is3-[[[6-Deoxy-4-O-(3,5-dichloro-2-ethyl-4,6-dihydroxybenzoyl)-2-O-methyl-β-D-mannopyranosyl]oxy]-methyl]-12(R)-[[6-deoxy-5-C-methyl-4-O-(2-methyl-1-oxopropyl)-β-D-lyxo-hexopyranosyl]oxy]-11(S)-ethyl-8(S)-hydroxy-18(S)-(1(R)-hydroxyethyl)-9,13,15-trimethyloxacyclooctadeca-3,5,9,13,15-pentaene-2-one.

Example 2 Analytical Data of OPT-80 and Related Substances

The analytical data of OPT-80 (which is composed almost entirely of theR-Tiacumicin B, which is the most active component of OPT-80) and threerelated compounds (S-Tiacumicin B, Lipiarmycin A4, and C-19 ketone) aresummarized below. The structures of these compounds are shown in FormulaVIII and Table 2 below.

TABLE 2 Structure of R-Tiacumicin B (the major most active component ofOPT-80) and related substances Compound X Y Z R-Tiacumicin B Ethyl(R)—OH Isopropyl S-Tiacumicin B Ethyl (S)—OH Isopropyl Lipiarmycin A4Methyl (S)—OH Isopropyl C-19 Ketone Ethyl ═O Isopropyl

Analytical Data of R-Tiacumicin B

mp 166-169° C. (white needle from isopropanol);

[α]_(D) ²⁰-6.9 (c 2.0, MeOH);

MS m/z (ESI) 1079.7 (M+Na)⁺;

¹H ¹H NMR NMR (400 MHz, CD₃OD) δ 7.21 (d, 1H), 6.59 (dd, 1H), 5.95 (ddd,1H), 5.83 (br s, 1H), 5.57 (t, 1H), 5.13 (br d, 1H), 5.09 (t, 1H), 5.02(d, 1H), 4.71 (m, 1H), 4.71 (br s, 1H), 4.64 (br s, 1H), 4.61 (d, 1H),4.42 (d, 1H), 4.23 (m, 1H), 4.02 (pentet, 1H), 3.92 (dd, 1H), 3.73 (m,2H), 3.70 (d, 1H), 3.56 (s, 3H), 3.52-3.56 (m, 2H), 2.92 (m, 2H),2.64-2.76 (m, 3H), 2.59 (heptet, 1H), 2.49 (ddd, 1H), 2.42 (ddd, 1H),2.01 (dq, 1H), 1.81 (s, 3H), 1.76 (s, 3H), 1.65 (s, 3H), 1.35 (d, 3H),1.29 (m, 1H), 1.20 (t, 3H), 1.19 (d, 3H), 1.17 (d, 3H), 1.16 (d, 3H),1.14 (s, 3H), 1.12 (s, 3H), 0.87 (t, 3H);

¹³C NMR (100 MHz, CD₃OD) δ 178.4, 169.7, 169.1, 154.6, 153.9, 146.2,143.7, 141.9, 137.1, 137.0, 136.4, 134.6, 128.5, 126.9, 125.6, 124.6,114.8, 112.8, 108.8, 102.3, 97.2, 94.3, 82.5, 78.6, 76.9, 75.9, 74.5,73.5, 73.2, 72.8, 71.6, 70.5, 68.3, 63.9, 62.2, 42.5, 37.3, 35.4, 28.7,28.3, 26.9, 26.4, 20.3, 19.6, 19.2, 18.7, 18.2, 17.6, 15.5, 14.6, 14.0,11.4.

Analytical Data of the S-Tiacumicin B

NaBH₄ (9 eq, 48 mg) was added in three portions to a solution of C-19Ketone (150 mg) in 3 mL MeOH. After 1 h, saturated NH₄Cl solution wasadded. The mixture was extracted with CHCl₃, and then concentrated.S-Tiacumicin B was purified by YMC-pack ODS-A 75×30 mm I.D. column(H₂O:MeOH:AcOH 28:72:1) yielding pure 35 mg of pure S-Tiacumicin B.

MS m/z 1074.5 (M+NH₄)⁺;

¹H NMR (400 MHz, CDCl₃) δ 7.15 (d, J=11.4 Hz, 1H), 6.58 (dd, J=14.1,11.4 Hz, 1H), 5.82 (ddd, J=14.1, 10.6, 3.5 Hz, 1H), 5.78 (s, 1H), 5.40(dd, J=7.8, 7.8 Hz, 1H), 5.15 (dd, J=9.5, 9.5 Hz, 1H), 5.01 (d, J=9.9Hz, 1H), 5.01 (d, J=9.9 Hz, 1H), 4.77 (ddd, J=5.8, 5.3, 5.3 Hz, 1H),4.68 (d, J=11.6 Hz, 1H), 4.65 (br s, 1H), 4.62 (br s, 1H), 4.42 (d,J=11.6 Hz, 1H), 4.28 (br s, 1H), 4.07-3.97 (m, 2H), 3.74-3.58 (m, 4H),3.61 (s, 3H), 3.52 (dq, J=9.5, 5.8 Hz, 1H), 3.08 (dq, J=12.6, 6.1 Hz,1H), 3.01 (dq, J=12.6, 6.1 Hz, 1H), 2.77-2.65 (m, 2H), 2.60 (heptet,J=6.9 Hz, 1H), 2.55-2.44 (m, 3H), 1.95-1.84 (m, 1H), 1.80 (s, 3H), 1.76(s, 3H), 1.66 (s, 3H), 1.34 (d, J=5.8 Hz, 3H), 1.29-1.24 (m, 1H), 1.27(d, J=6.6 Hz, 3H), 1.21 (t, J=6.1 Hz, 3H), 1.19 (d, J=6.9 Hz, 3H), 1.18(d, J=6.9 Hz, 3H), 1.15 (s, 3H), 1.10 (s, 3H), 0.84 (t, J=7.2 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃) δ 177.4, 170.1, 168.8, 157.6, 152.8, 144.4,143.1, 141.1, 136.7, 136.2, 134.9, 133.8, 128.7, 125.7, 125.2, 123.0,113.9, 107.5, 107.2, 101.7, 94.9, 92.6, 80.8, 79.2, 76.6, 74.8, 73.5,72.7, 71.9, 71.7, 70.2, 70.1, 69.5, 63.5, 62.3, 41.5, 36.6, 34.3, 29.5,28.2, 26.2, 26.0, 19.4, 19.3, 18.9, 18.5, 17.8, 17.3, 15.3, 14.1, 13.7,11.1;

Analytical data of Lipiarmycin A₄

MS m/z 1060.5 (M+NH₄)⁺;

¹H NMR (400 MHz, CDCl₃) δ 7.12 (d, J=11.6 Hz, 1H), 6.59 (dd, J=14.1,11.6 Hz, 1H), 5.85 (br s, 1H), 5.83 (ddd, J=14.1, 10.6, 4.8 Hz, 1H),5.47 (dd, J=8.3, 8.3 Hz, 1H), 5.12 (dd, J=9.6, 9.6 Hz, 1H), 5.00 (d,J=10.1 Hz, 1H), 4.98 (br d, J=10.6 Hz, 1H), 4.75-4.69 (m, 1H), 4.68 (d,J=11.4 Hz, 1H), 4.66 (br s, 1H), 4.62 (br s, 1H), 4.40 (d, J=11.4 Hz,1H), 4.26 (br s, 1H), 4.07-4.00 (m, 1H), 4.02 (br d, J=3.3 Hz, 1H),3.75-3.61 (m, 4H), 3.62 (s, 3H), 3.55 (dq, J=9.6, 6.1 Hz, 1H), 2.82-2.45(m, 6H), 2.60 (s, 3H), 2.07-1.97 (m, 1H), 1.92 (s, 3H), 1.81 (s, 3H),1.67 (s, 3H), 1.32 (d, J=6.1 Hz, 3H), 1.30-1.22 (m, 1H), 1.21 (d, J=6.6Hz, 3H), 1.19 (d, J=7.1 Hz, 3H), 1.18 (d, J=7.1 Hz, 3H), 1.15 (s, 3H),1.10 (s, 3H), 0.83 (t, J=7.2 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃) δ 177.4, 170.5, 168.9, 157.8, 153.0, 144.3,140.9, 137.7, 137.0, 136.3, 134.6, 134.4, 129.1, 127.9, 125.3, 123.2,114.5, 107.4, 107.0, 101.8, 94.7, 92.5, 80.3, 79.6, 76.7, 74.9, 73.5,72.7, 71.9, 71.6, 70.2, 70.1, 69.1, 63.6, 62.3, 41.9, 36.9, 34.4, 28.8,28.2, 25.9, 20.0, 19.3, 19.0, 18.6, 18.5, 17.8, 17.2, 15.5, 13.8. 11.2;

Analytical Data of C-19 Ketone

MS m/z 1072.5 (M+NH₄)⁺;

¹H NMR (400 MHz, CDCl₃) δ 7.27 (d, J=11.4 Hz, 1H), 6.61 (dd, J=14.7,11.4 Hz, 1H), 5.91 (ddd, J=14.7, 9.1, 5.8 Hz, 1H), 5.83 (s, 1H), 5.31(dd, J=7.9, 7.9 Hz, 1H), 5.14 (dd, J=9.7, 9.7 Hz, 1H), 5.06 (d, J=10.6Hz, 1H), 5.00 (d, J=10.1 Hz, 1H), 4.98 (dd, J=7.1, 4.8 Hz, 1H), 4.67 (d,J=11.9 Hz, 1H), 4.66 (br s, 1H), 4.61 (br s, 1H), 4.42 (d, J=11.9 Hz,1H), 4.30 (br s, 1H), 4.02 (br d, J=3.3 Hz, 1H), 3.63-3.60 (m, 4H), 3.62(s, 3H), 3.51 (dq, J=9.7, 6.1 Hz, 1H), 3.09 (dq, J=14.4, 7.3 Hz, 1H),3.03 (dq, J=14.4, 7.3 Hz, 1H), 2.76-2.50 (m, 6H), 2.21 (s, 3H),1.93-1.87 (m, 1H), 1.87 (s, 3H), 1.75 (s, 3H), 1.63 (s, 3H), 1.32 (d,J=6.1 Hz, 3H), 1.27-1.22 (m, 1H), 1.21 (t, J=7.3 Hz, 3H), 1.19 (d, J=7.1Hz, 3H), 1.18 (d, J=7.1 Hz, 3H), 1.14 (s, 3H), 1.10 (s, 3H), 0.84 (t,J=7.3 Hz, 3H);

¹³C NMR (100 MHz, CDCl₃) δ 205.5, 177.4, 170.1, 166.9, 157.6, 152.8,145.7, 143.1, 142.0, 137.1, 136.8, 135.5, 133.7, 128.3, 124.8, 124.0,122.8, 113.9, 107.3, 107.2, 101.3, 94.8, 92.4, 80.4, 77.7, 76.6, 74.7,73.5, 72.6, 71.8, 71.7, 70.2, 70.0, 63.0, 62.3, 41.5, 36.5, 34.3, 29.6,28.1, 26.2, 26.1, 26.0, 19.2, 18.9, 18.5, 17.8, 17.3, 15.2, 14.0, 13.3,11.0

Example 3 Biological Activity MIC Values Determined for Several C.difficile Strains

OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and itsrelated compounds were tested against C. difficile. The MIC values arereported below in Table 3. OPT-80 was surprisingly active when comparedto its enantiomer S-Tiacumicin B and Lipiarmycin A4.

TABLE 3 MIC (μg/ml) versus C. difficile strains R-Tiacumicin B (>90% C.difficile Stereomerically S-Tiacumicin Lipiarmycin C-19 strains Pure) BA4 Ketone ATCC 9689 0.03 0.125 0.06  0.06 ATCC 43255 0.125 1 0.5 0.5ATCC 17857 0.03 0.25 0.06 nd LC # 1 0.125 1 0.5 0.5 (Clinical isolate)

MIC Values Determined for Various Microorganisms

OPT-80 (which is composed almost entirely of the R-Tiacumicin B) and itsrelated compounds were tested against several other pathogens. The MICvalues are reported below in Table 4. OPT-80 was suprisingly active whencompared to S-Tiacumicin B and Lipiarmycin A4.

TABLE 4 MIC (μg/ml) against other microorganisms R-Tiacumicin B (>90%Strain Stereomerically Lipiarmycin ID # Organism Pure) S-Tiacumicin B A41 S. aureus 4 64 8 (ATCC 29213) 2 S. aureus, 4 64 16 (MRSA) 3 S. aureus,4 64 8 (MRSA) 4 E. faecalis 2 8 2 (ATCC 29212) 5 E. faecalis 4 32 16Vanc. resistant 6 E. faecalis 1 16 4 Vanc. resistant 7 E. faecium 1 8 4Vanc. resistant 8 E. faecium 1 32 32 Vanc. resistant

Example 4 Post-Antibiotic Effect of OPT-80 in C. difficile

The post-antibiotic effect (PAE) of OPT-80 (which is composed almostentirely of the R-Tiacumicin B) was measured versus two strains of C.difficile, ATCC 43255 and a clinical isolate, LC3. Vancomycin andrifampin were tested additionally versus LC3.

The PAE at 4× the MIC was observed to be extremely long: greater than 24hours, for both strains. Because of the long duration of this effect, anexact PAE was not calculated. Vancomycin, on the other hand, had a morenormal PAE of less than an hour when used at 4× the MIC versus strainLC3.

Example 5 In Vitro Activity of OPT-80

The in vitro efficacy of OPT-80 (which is composed almost entirely ofthe R-Tiacumicin B), metronidazole, and vancomycin were assessed versus110 genetically distinct clinical isolates of C. difficile via agardilution. The MIC data are presented in Tables 5 and 6.

TABLE 5 Geometric mean, MIC ranges, MIC₅₀, and MIC₉₀ values forOPT-80against 110 C. difficile clinical isolates, vancomycin, andmetronidazole, in μg/mL. Range Geometric Mean MIC₅₀ MIC₉₀ OPT-800.015-0.25 0.08 0.125 0.125 Metronidazole 0.025-0.5  0.15 0.125 0.25Vancomycin 0.06-4   0.8 1 1

TABLE 6 Raw MIC data for OPT-80, vancomycin (VAN), and metronidazole(MTZ) versus 110 clinical isolates of C. difficile, in μg/mL.R-Tiacumicin B (>90% ORG ID Stereomerically Pure) MTZ VAN A1 1535 0.1250.25 1 B1 832 0.06 0.125 1 D1 1360 0.03 0.25 1 E1 816 0.06 0.125 1 F11015 0.125 0.125 1 G1 1077 0.125 0.125 1 I1 1389 0.125 0.125 1 J1 59710.06 0.25 1 J7 4224 0.03 0.125 1 J9 4478 0.06 0.125 1 K1 4305 0.125 0.250.5 K14 5780 0.125 0.125 1 L1 1423 0.125 0.125 0.5 N1 471 0.125 0.1250.5 O1 1861 0.06 0.125 1 R1 397 0.125 0.125 1 R6 6015 0.015 0.25 2 V11521 0.125 0.125 0.5 W1 3931 0.125 0.5 1 X1 1890 0.125 0.125 1 Y1 56390.06 0.125 0.5 Y2 1459 0.06 0.125 1 Z1 3036 0.03 0.125 1 AA2 4380 0.0150.125 1 AB2 1725 0.06 0.125 1 AC1 1546 0.06 0.125 1 AF1 1808 0.125 0.1250.5 AG1 3044 0.125 0.125 1 AH1 3430 0.125 0.25 0.5 AJ1 1557 0.06 0.125 1AL1 1753 0.06 0.125 0.5 AN1 464 0.125 0.125 0.5 AO1 287 0.125 0.125 1AS1 4099 0.125 0.125 1 AT1 1216 0.125 0.125 1 AV1 941 0.25 0.125 0.5 CJ1893 0.125 0.025 1 AW1 4501 0.125 0.125 1 BE1 4307 0.125 0.25 1 BH1 45060.06 0.06 0.5 BI1 1675 0.125 0.125 1 BK1 4291 0.125 0.125 0.5 BL1 7160.125 0.125 1 BM1 1453 0.06 0.125 1 BN1 1322 0.125 0.25 1 BR1 1321 0.060.125 1 BT1 706 0.06 0.125 1 BV1 1183 0.125 0.25 1 BW1 3130 0.125 0.1251 BX1 4271 0.125 0.25 1 CN1 667 0.25 0.25 1 CB1 1584 0.25 0.125 1 CF15922 0.125 0.125 1 CG1 1566 0.125 0.125 1 CL1 3851 0.25 0.125 1 CO1 46520.25 0.125 1 CP1 5491 0.125 0.25 1 61 5930 0.03 0.25 1 63 6029 0.25 0.250.06 64 5940 0.125 0.25 1 65 5967 0.06 0.25 0.5 66 6366 0.015 0.125 0.567 6367 0.125 0.25 1 68 6368 0.03 0.125 0.06 69 6370 0.25 0.25 0.5 706376 0.125 0.25 2 71 6379 0.125 0.25 1 72 6380 0.125 0.25 2 73 6382 0.250.25 1 75 6388 0.125 0.125 0.5 76 6389 0.125 0.25 0.5 77 6390 0.06 0.1251 78 6392 0.015 0.03 0.5 80 6327 0.125 0.125 0.5 81 6328 0.125 0.125 0.582 6329 0.06 0.03 0.5 83 6330 0.06 0.125 0.5 84 6331 0.125 0.25 0.5 856332 0.06 0.125 1 86 6333 0.03 0.125 0.5 87 6334 0.125 0.125 0.5 88 63350.125 0.25 0.5 89 6336 0.25 0.5 1 90 6338 0.125 0.125 1 91 6339 0.1250.125 1 93 6341 0.125 0.125 1 94 6343 0.015 0.06 0.5 95 6347 0.125 0.1251 96 6348 0.06 0.125 0.5 97 6349 0.25 0.125 1 98 6350 0.125 0.5 1 1016354 0.015 0.06 1 102 6355 0.016 0.125 1 103 6068 0.06 0.125 1 104 60600.03 0.25 1 105 6071 0.03 0.125 0.5 106 6078 0.03 0.25 0.5 107 6079 0.060.125 0.5 109 6274 0.015 0.125 1 111 6279 0.03 0.125 1 112 6280 0.060.125 0.5 113 6304 0.06 0.125 1 114 386 0.06 0.125 4 115 5985 0.015 0.252 116 5702 0.06 0.125 1 117 6026 0.06 0.125 2 120 6057 0.03 0.25 1 1216072 0.06 0.25 0.5 122 6111 0.25 0.25 0.5 100 6353 0.125 0.25 1

Example 6 Activity of OPT-80 Compared Against Selected Anaerobic Species

The in vitro activity of OPT-80 was determined against 350 anaerobes.The experimental procedure for which is outlined in Antimicrobial Agentsand Chemotherapy, 2004, 48: 4430-4434, which is hereby incorporated byreference in its entirety.

All organisms, including the 21 C. difficile strains, were separateisolates and not clonally related. All quality-control gram-negative and-positive strains recommended by NCCLS were included with each run: inevery case, results (where available) were in range.

Results of MIC testing are presented in Table 7.

TABLE 7 MICs (μg/ml) of R-Tiacumicin B (>90% Stereomerically Pure)Organism MIC range MIC₅₀ MIC₉₀ Bacteroides fragilis (19)  64->128 >128 >128 Non-fragilis B. fragilis group   64->128 >128 >128species (38) Prevotella/Porphyromonas species   16->128 >128 >128 (42)Fusobacterium nucleatum (14)   64->128 >128 >128 Fusobacteriummortiferum (10)   64->128 >128 >128 Fusobacterium species,  16->128 >128 >128 miscellaneous (14) Peptostreptococcus tetradius (16) 0.25-2.0 1.0 1.0 Peptostreptococcus  0.25-1.0 0.5 1.0 asaccharolyticus(15) Peptostreptococcus anaerobius <0.016-0.03 <0.016 <0.016 (15)Finegoldia magna (15)  0.25-2.0 1.0 1.0 Micromonas micros (14)<0.016-0.06 0.03 0.06 Peptostreptococcus prevotii (3)  0.25-1.0 NA NAPropionibacterium acnes (20)  0.5-1.0 4.0 4.0 Eggerthella lenta (10)<0.016-0.06 <0.016 <0.03 Miscellaneous gram-positive <0.016-16   <0.12516 non-spore-forming rods (20) Clostridium perfringens (35) <0.016-0.06<0.016 0.03 Clostridium difficile (21) <0.016-0.25 <0.016 0.125Clostridium tertium (10) <0.016-0.06 <0.016 0.03 Clostridium species(19) <0.016-0.06 <0.016 0.03 Clostridium spp. (all) (85) <0.016-0.06<0.016 0.06

Example 7 In Vitro Activities of OPT-80 Against Intestinal Bacteria

The in vitro activity of OPT-80 against intestinal bacteria wasevaluated. The experimental procedure for which is outlined inAntimicrobial Agents and Chemotherapy, 2004, 48: 4898-4902, which ishereby incorporated by reference in its entirety.

Antimicrobial concentration ranges were selected to encompass or surpassthe levels that would be achieved in the gut (to the extent that thisinformation is available), subject to the limitations of solubility ofthe drugs in the testing medium. The range of concentration of OPT-80used during testing was 0.03 μg/ml to 1024 μg/ml.

For analysis, the bacteria tested were generally placed into genus,species, or other groups with at least 10 isolates. The ranges and theMICs at which 50 and 90% of isolates were inhibited were determinedexcept for organisms with fewer than 10 strains tested, for which onlythe ranges are reported (Table 8).

OPT-80 had good activity against most anaerobic gram-positivenon-spore-forming rods and anaerobic gram-positive cocci. OPT-80 alsoshowed good activity against enterococci and staphylococci.

TABLE 8 In vitro activity of R-Tiacumicin B (>90% Stereomerically Pure)against 453 bacterial isolates Organism MIC range MIC₅₀ MIC₉₀Bacteroides fragilis group spp.  256->1024 256 >1024 (50) Veillonellaspp. (10) 16-128  32 128 Other anaerobic gram-negative 0.06-1024  1024 >1024 rods (51) All anaerobic gram-negative 0.06->1024 256 >1024species (111) Clostridium bifermentans (9) 0.06 NA NA Clostridiumbolteae (7) 1-64  NA NA Clostridium clostridioforme (4)  4-128 NA NAClostridium difficile (23) 0.06-2   0.12 0.25 Clostridium glycolicum (9)0.06-1   NA NA Clostridium innocuum (9) 32-128  NA NA Clostridiumparaputrificum (8) 0.06-8   NA NA Clostridium perfringens (14) 0.060.062 0.062 Clostridium ramosum (10) 256-512   512 512 Clostridiumsordellii (5) 0.06 NA NA Other clostridial species (9) 0.06->1024 NA NAAll Clostridium species (107) 0.06->1024 0.062 128 Anaerobicnon-spore-forming 0.06->1024 1 32 gram-positive rods (63) Anaerobicgram-positive cocci (49) 0.06->1024 0.5 2 All anaerobic gram-positive0.06->1024 0.12 64 species (219) Streptococcus, formerly S. milleri16-64   32 32 group (14) Other Streptococcus species (9) 16-128  NA NAEnterococcus species (21) 2.0-16  8 8 Staphylococcus aureus and 0.25-2  0.5 2 Staphylococcus epidermidis (19) Total for all strains (453)0.06->1024 8 1024

Other Embodiments

All references discussed above are herein incorporated by reference intheir entirety for all purposes. While this invention has beenparticularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the spirit and scope of the invention as defined by theappended claims.

1-16. (canceled)
 17. A method of treating reoccurring diarrhea caused byC. difficile gastrointestinal infection in a human patient previouslytreated for the diarrhea, comprising orally administering to saidpatient a therapeutically effective amount of a compound having theformula (IV):

or a pharmaceutically acceptable salt combined with one or morepharmaceutically acceptable carriers, wherein the compound havingformula (IV) is greater than 90% by weight stereomerically pure.
 18. Themethod of claim 17, wherein the method consists of administering to thehuman patient a therapeutically effective amount of the compound havingformula (IV) or a pharmaceutically acceptable salt thereof combined withone or more pharmaceutically acceptable carriers.
 19. The method ofclaim 17, wherein the compound of formula (IV) is formulated as atablet.
 20. The method of claim 17, wherein the compound of formula (IV)is formulated as a capsule.
 21. The method of claim 17, wherein thecompound of formula (IV) is greater than 93% by weight stereomericallypure.
 22. The method of claim 17, wherein the compound of formula (IV)is greater than 95% by weight stereomerically pure.
 23. The method ofclaim 17, wherein the compound of formula (IV) is greater than 97% byweight stereomerically pure.
 24. The method of claim 17, wherein thecompound of formula (IV) is substantially free of other diastereomers ofthe compound.
 25. The method of claim 17, wherein the patient suffersfrom colitis.
 26. A method of treating reoccurring diarrhea caused by C.difficile gastrointestinal infection in a human patient previouslytreated for the diarrhea, consisting of orally administering to saidpatient a therapeutically effective amount of a compound having theformula (IV):

or a pharmaceutically acceptable salt thereof combined with one or morepharmaceutically acceptable carriers, wherein the compound of theformula (IV) is greater than 93% by weight stereomerically pure.
 27. Themethod of claim 26, wherein the compound of formula (IV) is formulatedas a tablet.
 28. The method of claim 26, wherein the compound isformulated as a capsule.
 29. The method of claim 26, wherein thecompound of formula (IV) is greater than 95% by weight stereomericallypure.
 30. The method of claim 26, wherein the compound of formula (IV)is greater than 97% by weight stereomerically pure.
 31. The method ofclaim 26, wherein the compound of formula (IV) is substantially free ofother diastereomers of the compound.
 32. The method of claim 26, whereinthe patient suffers from colitis.